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Analysis of the TNFα-dependent
Apoptosis - Survival Switch in Hepatocytes
by a Systems Biology Approach




INAUGURALDISSERTATION
zur Erlangung des Doktorgrades
der Fakultät für Chemie, Pharmazie und Geowissenschaften
der Albert-Ludwigs-Universität
Freiburg im Breisgau



vorgelegt von

Kathrin Schmich
aus Schweinfurt

November 2010 Dekan: Prof. Dr. Harald Hillebrecht
Leiterin der Arbeit: Prof. Dr. Irmgard Merfort
Referentin: Prof. Dr. Irmgard Merfort
Korreferentin: Prof. Dr. Regine Süss
Drittprüfer: Prof. Dr. Christoph Borner




Tag der Verkündigung des Prüfungsergebnisses: 03.02.2011






Parts of this work have been published in the following conference presentations and
publications:




Publications

K. Naumann, K. Schmich, C. Jaeger, I.Merfort
Noxa/Mcl-1 balance decides the effectiveness of MG132 with chemotherapeutics
in pancreatic cancer
In preparation

* * † †R. Schlatter , K. Schmich , J. Trefzger, O. Sawodny, M. Ederer , I. Merfort
†(* Both authors contributed equally to this work, shared senior authorship)
Modeling the TNFα-induced apoptosis pathway in hepatocytes
Submitted

* *K. Schmich, R. Schlatter , N. Corazza , K. Sá Ferreira, M. Ederer, T. Brunner,
C. Borner, I. Merfort
(* Both authors contributed equally to this work)
TNFα sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in
a Bim- and Bid-dependent manner
Hepatology, 2010, Sept 24, ePub

* *T. Sparna, J. Rétey , K. Schmich , U. Albrecht, K. Naumann, N. Gretz, H.P. Fischer,
J.G. Bode, I. Merfort
(* Both authors contributed equally to this work)
Genome-wide comparison between IL-17 and combined TNF-alpha/ IL-17
induced genes in primary murine hepatocytes
BMC Genomics. 2010 Apr 7;11:226

R. Schlatter, K. Schmich, I. Avalos Vizcarra, P. Scheurich, T. Sauter, C. Borner,
M. Ederer, I. Merfort Sawodny O.
ON/OFF and beyond-a boolean model of apoptosis
PLoS Computational Biology, 2009 Dec;5(12):e1000595. Epub

M. Misiek, J. Williams, K. Schmich, W. Hüttel, I. Merfort, C.E. Salomon,
C.C. Aldrich, D. Hoffmeister
Structure and cytotoxicity of arnamial and related fungal sesquiterpene aryl
esters.
Journal of Natural Products, 2009 Oct;72(10):1888-91.

D. Walter, K. Schmich, S. Vogel, R. Pick, T. Kaufmann, F.C. Hochmuth, A. Haber,
K. Neubert, S. MacNelly, F. von Weizsäcker, I. Merfort, U. Maurer, A. Strasser,
C. Borner
Switch from type II to type I Fas/CD95 death signaling upon in vitro culturing
of primary hepatocytes
Hepatology, 2008, Vol. 48, No. 6, 1942-1953



Conference talks


K. Schmich, R. Schlatter, T. Sauter, C. Borner, M. Ederer, O. Sawodny, I. Merfort
ON/OFF and beyond - a comprehensive Boolean model of apoptosis
Hepatosys Verbundtreffen Munzingen, October 2009

K. Schmich
Vom Giftmischer zum modernen Wirkstoffforscher
Kepler Seminar Stuttgart, October 2009

K. Schmich, R. Schlatter, M. Ederer, J. Timmer, O. Sawodny, C. Borner, I. Merfort
Crosstalk between Fas/CD95-induced apoptosis and TNF-α in primary mouse
hepatocytes: A systems biology approach
German Symposium on Systems Biology, Heidelberg, May, 2009

K. Schmich, D. Walter, R. Schlatter, F. v. Weizsäcker, T. Sauter, O. Sawodny,
J. Timmer, C. Borner, I. Merfort
TNF-α sensitizes primary mouse hepatocytes to Fas/CD95-induced apoptosis: A
systems biology approach
th
12 International TNF Conference, San Lorenzo de El Escorial, Madrid, April, 2009

K. Schmich
Modeling of the NF-κB-dependent apoptosis/survival switch: A systems biology
approach
Doktorandenseminar December 2008

K. Schmich, C. Borner, R. Schlatter, D. Walter, O. Sawodny, I. Merfort
TNF-α sensitizes hepatocytes to Fas/CD95-induced apoptosis
Hepatosys Verbundtreffen Munzingen October 2008



Conference posters


R. Schlatter, K. Schmich, , O. Sawodny, M. Ederer, I. Merfort
Modeling of TNFα induced apoptosis in primary mouse hepatocytes: A dynamic
view on cell death execution
th 11 International Conference on Systems Biology (ICSB), Edinburgh, Scotland,
October 2010

R. Schlatter, K. Schmich, I. Merfort, O. Sawodny, M. Ederer
A large-scale logical model of apoptosis: experimental validation, analysis,
development of quality assurance methods, model fusion and definition of
model standards
th 11 International Conference on Systems Biology (ICSB), Edinburgh, Scotland,
October 2010

K. Schmich, R. Schlatter, J. Trefzger, C. Borner, M. Ederer, I. Merfort
Modeling TNF-α induced apoptosis: A dynamic view on cell death execution
th
7 European workshop on cell death (EWCD), Tisvildeleje, Denmark, July 2010

K. Schmich, R. Schlatter, J. Trefzger, C. Borner, M. Ederer, I. Merfort
Modeling of TNF-α induced apoptosis in primary mouse hepatocytes: A
dynamic view on cell death execution
Conference on Systems Biology of Mammalian Cells (SBMC) 2010, Freiburg, June
2010

R. Schlatter, K. Schmich, I. A. Vizcarra, T. Sauter, C. Borner, M. Ederer, I. Merfort,
O. Sawodny
ON/OFF – A Boolean Model of Apoptosis
Cancer Systems Biology - Molecular Mechanisms and Mathematical Modeling,
Rostock, June 2009


R. Schlatter, I. Avalos Vizcarra, K. Schmich, T. Sauter, M. Ederer, I. Merfort, O.
Sawodny
ON/OFF- A Boolean Model of Apoptosis
German Symposium on Systems Biology 2009, Heidelberg, May 2009

K. Schmich, R.Schlatter, D. Walter, T.Sparna, U.Albrecht, A. Kulawik, S. MacNelly,
F. von Weizsäcker, T. Sauter, O. Sawodny, K. Bartholomé, J. Timmer, J. Bode,
C. Borner, I. Merfort
TNF-α sensitizes primary hepatocytes to Fas/CD95-induced apoptosis: A
systems biology approach
Evaluation of the competence network HepatoSys, Berlin, January 2009

T. Sparna, J. Rétey, K. Schmich, U. Albrecht, A. Kulawik, F. Schliess, H.P. Fischer,
W. Schmidt-Heck, R. Gutke, S. MacNelly, F. v. Weizsäcker, R. Nitschke,
K. Bartholomé, J. Timmer, J. Bode, I. Merfort
Cytokine induced gene expression in hepatocytes reveals common and distinct
target gene subsets
Evaluation of the competence network HepatoSys, Berlin, January 2009

K. Schmich, D. Walter, R. Schlatter, T. Sparna, S. MacNelly, F. von Weizsäcker,
T. Sauter, O. Sawodny, K. Bartholomé, J. Timmer, C. Borner and I. Merfort
TNF-α sensitizes primary mouse hepatocytes to Fas/CD95-induced apoptosis
Conference on Systems Biology of Mammalian Cells (SBMC) 2008, Dresden, May
2008

D. Walter, S. Vogel, K. Schmich, R. Pick, T. Kaufmann, F.C. Hochmuth,
R. Nitschke, A. Haber, K. Neubert, S. MacNelly, F.v. Weizsäcker, I. Merfort, J.
Silke, A. Strasser, C. Borner
Switch from type II to I Fas/CD95 death signaling upon in vitro culturing of
primary mouse hepatocytes
Conference on Systems Biology of Mammalian Cells (SBMC) 2008, Dresden, May
2008

K. Schmich, T. Sparna, U. Albrecht, J. Retéy, H.P. Fischer, K. Bartholomé,
J. Timmer, S. MacNelly, F. v. Weizsäcker, R. Nitschke , C. Borner, J. Bode, I.
Merfort
Modeling of the NF-kB dependent apoptosis/regeneration switch in hepatocytes
Statusseminar BMBF, Heidelberg, October 2007






















Table of Contents I
1. Introduction......................................................................................................... 1
1.1. Liver regeneration and pathophysiology............................................................ 1
1.1.1. Cytokines and groth factors in liver regeneration ...........................................................1
1.1.2. Mechanisms of liver injury .............................................................................................4
1.2. Apoptosis ............................................................................................................... 6
1.2.1. Type I and type II apoptosis pathway .............................................................................8
1.2.2. Bcl-2 protein family and the mitochondrial apoptosis pathway....................................10
1.2.3. Fas (CD95) signaling in the liver ..................................................................................13
1.2.4. TNFα mediated apoptosis .............................................................................................14
1.3. Systems biology of apoptosis.............................................................................. 18
1.4. Aims of this work................................................................................................ 21
2. Results ............................................................................................................... 23
2.1 Death receptor induced apoptosis in collagen cultured primary murine
hepatocytes ....................................................................................................................... 23
2.1.1. TNFα induces apoptosis in primary hepatocytes only under transcriptional arrest.......23
2.1.2. TNFα/ActD induces type II apoptosis pathway in primary murine hepatocytes...........25
2.1.3. ROS accumulation and sustained JNK activation are critical mediators of TNFα/ActD
apoptosis......................................................................................................................................28
2.1.4. The NF-κB target genes cFLIP and MnSOD control TNFα/ActD apoptosis................33
2.1.5. Mathematical modeling of the TNFα and actinomycin D induced apoptosis signaling36
2.2 Crosstalk of TNFα and FasL signaling in hepatocyte apoptosis .................... 38
2.2.1. TNFα sensitizes primary murine hepatocytes to Fas-induced apoptosis.......................39
2.2.2. TNFα sensitization is specific for TNFα.......................................................................42
2.2.3. TNFα directly interferes with Fas signaling without upregulating Fas or FasL............45
2.2.4. Bid but not XIAP is essential for the sensitizing effect ................................................46
2.2.5. TNFα activates JNK and its inhibition blocks the sensitization....................................51
2.2.6. Bim is crucial for the TNFα sensitizing effect ..............................................................54
2.2.7. Type II Fas signaling is restored upon TNFα sensitization...........................................57
2.2.8. Expression analysis of the important apoptosis modulators cFLIP, cIAP1 and 2, Bcl-xL
and Mcl-1 ....................................................................................................................................59
2.2.9. Mathematical modeling confirms the sensitizing effect................................................61
2.3 A logical model of apoptosis............................................................................... 64
2.3.1. Apoptosis induction by different stimuli in type I and type II cells..............................64
2.3.2. Regulation of the NF-κB activity and antiapoptotic target genes cIAP2 and cFLIP.....67
2.3.3. A Boolean model to simulate apoptosis and survival ...................................................70
2.4 Differential gene expression by TNFα, IL-1β and IL-17 in primary murine
hepatocytes ....................................................................................................................... 72 II Table of Contents
2.4.1. Activation of the transcription factor NF-κB................................................................ 72
2.4.2. Comparison between TNFα and TNFα/IL-17 induced genes....................................... 73
2.4.3. Time course analysis of cytokine target genes ............................................................. 76
2.4.4. Modulation of the IκB-ζ mRNA half life time by IL-17 .............................................. 78
3. Discussion ..........................................................................................................81
4. Summary ..........................................................................................................103
5. Materials and Methods....................................................................................107
5.1 Materials............................................................................................................ 107
5.1.1. Chemicals ................................................................................................................... 107
5.1.2. Chemicals and consumables for hepatocyte isolation................................................. 109
5.1.3. Cytokines, primers, enzymes, siRNA and kits ........................................................... 110
5.1.4. Mice............................................................................................................................ 111
5.1.5. Consumables............................................................................................................... 112
5.1.6. Equipment................................................................................................................... 113
5.2 Methods ............................................................................................................. 115
5.2.1. Cell culture ................................................................................................................. 115
5.2.2. Preparation of protein extracts.................................................................................... 120
5.2.3. Apoptosis and Cytotoxicity methods.......................................................................... 123
5.2.4. Methods for transcription factor binding studies ........................................................ 128
5.2.5. Gene expression methods ........................................................................................... 132
5.2.6. Gene knock-down methods ........................................................................................ 143
5.2.7. Protein detection methods........................................................................................... 145
5.2.8. Statistical analysis....................................................................................................... 151
6. Reference List ..................................................................................................153
7. List of Figures..................................................................................................165
8. List of Tables....................................................................................................169
9. List of Abbreviations........................................................................................171
10. Acknowledgement........................................................................................175
11. Curriculum Vitae.........................................................................................179

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